Microbead-based assays have evolved into powerful tools for the multiplex detection of biomolecules. Analytes are captured by DNA or protein capture molecules which are coupled on microbead surfaces. A homogeneous carboxylation of microbeads is essential for the optimal and reproducible coupling of capture molecules and thus a prerequisite for an optimal multiplex microbead-based assay performance. We developed a simple fluorescence dye adsorption assay for the description of microbead carboxylation and for the prediction of coupling successes of capture molecules. Using the fluorescence dye SYTO-62 it is possible to quantify the degree of carboxylation of poly(methyl methacrylate) (PMMA) microbeads within 1 h in a multiplex format by fluorescence microscopy or flow cytometry. Compared to conventional bulk assays which only provide an average degree of carboxylation the main advantage of the SYTO-62 assay is the single microbead analysis and therefore the description of the qualitative distribution of carboxylation in microbead populations. The SYTO-62 assay is sensitive enough to even determine weak carboxylation. Also, the quality of microbeads can be evaluated. To our knowledge this is the first report which applies a reversible noncovalent fluorescent dye adsorption assay to quantify the degree of carboxylation on surfaces.
The second affiliation for the tenth author is not indicated. Pier Luigi Meroni is also affiliated with: IRCCS Istituto Auxologico Italiano, Milan, Italy.
Abstract Introduction Indirect immunofluorescence (IIF) employing ethanol-fixed neutrophils (ethN) is still the method of choice for assessing antineutrophil cytoplasmic antibodies (ANCA) in ANCA-associated vasculitides (AAV). However, conventional fluorescence microscopy is subjective and prone to high variability. The objective of this study was to evaluate novel pattern recognition algorithms for the standardized automated interpretation of ANCA patterns. Methods Seventy ANCA-positive samples (20 antimyeloperoxidase ANCA, 50 antiproteinase3 ANCA) and 100 controls from healthy individuals analyzed on ethN and formalin-fixed neutrophils (formN) by IIF were used as a 'training set' for the development of pattern recognition algorithms. Sera from 342 patients ('test set') with AAV and other systemic rheumatic and infectious diseases were tested for ANCA patterns using the novel pattern recognition algorithms and conventional fluorescence microscopy. Results Interpretation software employing pattern recognition algorithms was developed enabling positive/negative discrimination and classification of cytoplasmic ANCA (C-ANCA) and perinuclear ANCA (P-ANCA). Comparison of visual reading of the 'test set' samples with automated interpretation revealed Cohen's kappa (κ) values of 0.955 on ethN and 0.929 on formN for positive/negative discrimination. Analysis of the 'test set' with regard to the discrimination between C-ANCA and P-ANCA patterns showed a high agreement for ethN (κ = 0.746) and formN (κ = 0.847). There was no significant difference between visual and automated interpretation regarding positive/negative discrimination on ethN and formN, as well as ANCA pattern recognition ( P > 0.05, respectively). Conclusions Pattern recognition algorithms can assist in the automated interpretation of ANCA IIF. Automated reading of ethN and formN IIF patterns demonstrated high consistency with visual ANCA assessment.
Abstract If there is a suspicion of a systemic autoimmune disease, a two-step assessment of autoantibodies (AAb) is recommended for the serological diagnosis thereof. First, AAb will be determined using sensitive, cell-based indirect immunofluorescence. Then, a positive result must be confirmed with a more specific test due to the possibility of false-positive results. This gradual approach is necessary because there is currently no assay technique that fulfills the requirements for a one-stage procedure for sensitivity and specificity. For effective AAb analysis, simultaneous determination of several AAb with multiparametric confirmatory assays significantly shortens serological diagnosis, compared with conventional monoparametric testing. Yet, currently available multiparametric AAb detection techniques do not offer the combination of screening and confirmatory testing. Thus, a new approach based on digital fluorescence was developed by applying a novel CytoBead technology that is presented here. The aim was to combine the recommended stepwise approach consisting of sensitive screening and confirmation of specific diagnosis in a reaction environment and thereafter the possibility of adaptation to the serological diagnosis of several autoimmune diseases. Using standard microscopic glass slides and the combination of native cellular or tissue substrates with autoantigen-loaded fluorescent microparticles (beads) in a reaction environment, along with the possibility of manual and automatic evaluation by IIF and the quantitative measurement of fluorescent signals, the disadvantages of currently existing test systems could be overcome. This novel concept is applicable for the determination of various multiparametric AAb, e.g., the determination of antinuclear antibodies and the corresponding AAb in molecular cytoplasmic and nuclear autoantigenic structures. Further, this becomes the basis for the simultaneous multiparametric AAb determination for the serology of celiac disease or ANCA-associated vasculitides.
Glycoprotein-2 (GP2) IgA is a predictor of disease severity in primary sclerosing cholangitis (PSC). We examined GP2′s occurrence in the biliary tract, the site of inflammation. GP2 was analyzed using ELISA, immunoblotting, mass spectrometry, and immunohistochemistry. The samples included: 20 bile and 30 serum samples from PSC patients, 23 bile and 11 serum samples from patients with gallstone disease (GD), 15 bile samples from healthy individuals undergoing liver-donation surgery (HILD), 20 extracts of gallstones (GE) obtained during cholecystectomy, and 101 blood-donor sera. Biliary GP2 concentrations were significantly higher in patients with PSC and GD than in HILD (p < 0.0001). Serum GP2 levels were similar in PSC and GD patients, and controls, but lower than in bile (p < 0.0001). GP2 was detected in all 20 GEs. Mass spectrometry identified GP2 in the bile of 2 randomly selected GD and 2 PSC patients, and in none of 2 HILD samples. GP2 was found in peribiliary glands in 8 out of 12 PSC patients, showing morphological changes in acinar cells, but not in GD-gallbladders. GP2 is present in bile of PSC and GD patients. It is synthesized in the peribiliary glands of PSC patients, supporting a pathogenic role for biliary GP2 in PSC.
Rapidly progressive glomerulonephritis (RPGN) is mainly caused by anti-glomerular basement membrane (GBM) antibody-mediated glomerulonephritis, immune-complex or anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides and leads to rapid loss of renal function. Detection of ANCA and autoantibodies (autoAbs) to GBM and dsDNA enables early diagnosis and appropriate treatment of RPGN aiding in preventing end-stage renal disease. Determination of ANCA on neutrophils (ANCA) as well as autoAbs to myeloperoxidase (MPO-ANCA), proteinase 3 (PR3-ANCA), GBM, and dsDNA was performed by the novel multiplex CytoBead technology combining cell- and microbead-based autoAb analyses by automated indirect immunofluorescence (IIF). Forty patients with granulomatosis with polyangiitis (GPA), 48 with microscopic polyangiitis (MPA), 2 with eosinophilic GPA, 42 with systemic lupus erythematosus (SLE), 43 with Goodpasture syndrome (GPS), 57 with infectious diseases (INF), and 55 healthy subjects (HS) were analyzed and findings compared with classical single testing. The CytoBead assay revealed for GPA, MPA, GPS, and SLE the following diagnostic sensitivities and for HS and INF the corresponding specificities: PR3-ANCA, 85.0% and 100.0%; MPO-ANCA, 77.1% and 99.1%; anti-GBM autoAb, 88.4% and 96.4%; anti-dsDNA autoAb, 83.3% and 97.3%; ANCA, 91.1% and 99.1%, respectively. Agreement with classical enzyme-linked immunosorbent assay and IIF was very good for anti-GBM autoAb, MPO-ANCA, PR3-ANCA, and ANCA, respectively. Anti-dsDNA autoAb comparative analysis demonstrated fair agreement only and a significant difference (P = 0.0001). The CytoBead technology provides a unique multiplex reaction environment for simultaneous RPGN-specific autoAb testing. CytoBead RPGN assay is a promising alternative to time-consuming single parameter analysis and, thus, is well suited for emergency situations.
